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1.
J Biol Chem ; 294(34): 12581-12598, 2019 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-31285263

RESUMEN

Patients with fatty liver diseases present altered mitochondrial morphology and impaired metabolic function. Mitochondrial dynamics and related cell function require the uncleaved form of the dynamin-like GTPase OPA1. Stabilization of OPA1 might then confer a protective mechanism against stress-induced tissue damages. To study the putative role of hepatic mitochondrial morphology in a sick liver, we expressed a cleavage-resistant long form of OPA1 (L-OPA1Δ) in the liver of a mouse model with mitochondrial liver dysfunction (i.e. the hepatocyte-specific prohibitin-2 knockout (Hep-Phb2-/-) mice). Liver prohibitin-2 deficiency caused excessive proteolytic cleavage of L-OPA1, mitochondrial fragmentation, and increased apoptosis. These molecular alterations were associated with lipid accumulation, abolished gluconeogenesis, and extensive liver damage. Such liver dysfunction was associated with severe hypoglycemia. In prohibitin-2 knockout mice, expression of L-OPA1Δ by in vivo adenovirus delivery restored the morphology but not the function of mitochondria in hepatocytes. In prohibitin-competent mice, elongation of liver mitochondria by expression of L-OPA1Δ resulted in excessive glucose production associated with increased mitochondrial respiration. In conclusion, mitochondrial dynamics participates in the control of hepatic glucose production.


Asunto(s)
GTP Fosfohidrolasas/metabolismo , Gluconeogénesis , Hepatocitos/metabolismo , Mitocondrias/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis , Respiración de la Célula , Hepatocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prohibitinas , Proteínas Represoras/deficiencia
2.
Glia ; 68(9): 1824-1839, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32092215

RESUMEN

AMP-activated protein kinase (AMPK) is an important energy sensor located in cells throughout the human body. From the periphery, AMPK is known to be a metabolic master switch controlling the use of energy fuels. The energy sensor is activated when the energy status of the cell is low, initiating energy-producing pathways and deactivating energy-consuming pathways. All brain cells are crucially dependent on energy production for survival, and the availability of energy substrates must be closely regulated. Intriguingly, the role of AMPK in the regulation of brain cell metabolism has been sparsely investigated, particularly in astrocytes. By investigating metabolism of 13 C-labeled energy substrates in acutely isolated hippocampal slices and cultured astrocytes, with subsequent mass spectrometry analysis, we here show that activation of AMPK increases glycolysis as well as the capacity of the TCA cycle, that is, anaplerosis, through the activity of pyruvate carboxylase (PC) in astrocytes. In addition, we demonstrate that AMPK activation leads to augmented astrocytic glutamate oxidation via pyruvate recycling (i.e., cataplerosis). This regulatory mechanism induced by AMPK activation is mediated via glutamate dehydrogenase (GDH) shown in a CNS-specific GDH knockout mouse. Collectively, these findings demonstrate that AMPK regulates TCA cycle dynamics in astrocytes via PC and GDH activity. AMPK functionality has been shown to be hampered in Alzheimer's and Parkinson's disease and our findings may therefore add to the toolbox for discovery of new metabolic drug targets.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Astrocitos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Astrocitos/metabolismo , Respiración de la Célula , Ciclo del Ácido Cítrico , Glutamato Deshidrogenasa , Ratones , Estrés Oxidativo
3.
Hum Mol Genet ; 26(18): 3453-3465, 2017 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-28911206

RESUMEN

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.


Asunto(s)
Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Adenosina Difosfato/metabolismo , Aminoácidos/genética , Animales , Glucemia/metabolismo , Hiperinsulinismo Congénito/genética , Glucosa/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperinsulinismo/genética , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Polimorfismo de Nucleótido Simple/genética
4.
Proc Natl Acad Sci U S A ; 113(33): E4847-56, 2016 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-27482114

RESUMEN

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state, but their role in inflammation is still unclear. Here, using confocal intravital microscopy, we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins, platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets, Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level, we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation.


Asunto(s)
Antígenos Ly/análisis , Proteína 61 Rica en Cisteína/fisiología , Monocitos/fisiología , Vasculitis/etiología , Animales , Plaquetas/fisiología , Movimiento Celular , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiología
5.
J Biol Chem ; 291(25): 13063-75, 2016 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-27137930

RESUMEN

Efficient energy storage in adipose tissues requires optimal function of the insulin-producing ß-cell, whereas its dysfunction promotes diabetes. The associated paradox related to ß-cell efficiency is that excessive accumulation of fat in adipose tissue predisposes for type 2 diabetes. Insulin exocytosis is regulated by intracellular metabolic signal transduction, with glutamate dehydrogenase playing a key role in the amplification of the secretory response. Here, we used mice with ß-cell-selective glutamate dehydrogenase deletion (ßGlud1(-/-)), lacking an amplifying pathway of insulin secretion. As opposed to control mice, ßGlud1(-/-) animals fed a high calorie diet maintained glucose tolerance and did not develop diet-induced obesity. Islets of ßGlud1(-/-) mice did not increase their secretory response upon high calorie feeding, as did islets of control mice. Inhibited adipose tissue expansion observed in knock-out mice correlated with lower expression of genes responsible for adipogenesis. Rather than being efficiently stored, lipids were consumed at a higher rate in ßGlud1(-/-) mice compared with controls, in particular during food intake periods. These results show that reduced ß-cell function prior to high calorie feeding prevented diet-induced obesity.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Células Secretoras de Insulina/fisiología , Obesidad/etiología , Transducción de Señal , Animales , Metabolismo Basal , Células Cultivadas , Intolerancia a la Glucosa , Glutamato Deshidrogenasa/genética , Metabolismo de los Lípidos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/patología
6.
Neurochem Res ; 39(3): 456-9, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-23595828

RESUMEN

In the brain, Glud1-encoded glutamate dehydrogenase plays a major role in the recycling of the neurotransmitter glutamate. We recently reported a new model of brain-specific Glud1 null mice (Cns-Glud1 (-/-)) lacking glutamate dehydrogenase in the central nervous system. Cns-Glud1 (-/-) mice exhibit reduced astrocytic glutamate breakdown and redirection of glutamate pathways without altering synaptic transmission. Cns-Glud1 (-/-) mice were generated using LoxP and Nestin-Cre technology. Nestin-Cre mice are widely used to investigate gene deletion in the central nervous system. However, the Nes-Cre transgene itself was reported to induce a phenotype related to body weight gain. Here, we review the potential side-effects of Nes-Cre and analysed Cns-Glud1 (-/-) body weight gain. Overall, Nestin-Cre mice may exhibit transient and moderate growth retardation during the few weeks immediately following weaning. Pending appropriate controls and homogenization of the genetic background, Nestin-Cre technology is a valuable tool enabling disruption of genes of interest in the central nervous system.


Asunto(s)
Encéfalo/metabolismo , Nestina/genética , Regiones Promotoras Genéticas , Receptores de Glutamato/genética , Eliminación de Secuencia , Animales , Glutamato Deshidrogenasa , Humanos , Integrasas/genética , Integrasas/metabolismo , Ratones
7.
Cell Metab ; 7(2): 113-24, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18249171

RESUMEN

JunD, a transcription factor of the AP-1 family, protects cells against oxidative stress. Here, we show that junD(-/-) mice exhibit features of premature aging and shortened life span. They also display persistent hypoglycemia due to enhanced insulin secretion. Consequently, the insulin/IGF-1 signaling pathways are constitutively stimulated, leading to inactivation of FoxO1, a positive regulator of longevity. Hyperinsulinemia most likely results from enhanced pancreatic islet vascularization owing to chronic oxidative stress. Indeed, accumulation of free radicals in beta cells enhances VEGF-A transcription, which in turn increases pancreatic angiogenesis and insulin secretion. Accordingly, long-term treatment with an antioxidant rescues the phenotype of junD(-/-) mice. Indeed, dietary antioxidant supplementation was protective against pancreatic angiogenesis, hyperinsulinemia, and subsequent activation of insulin signaling cascades in peripheral tissues. Taken together, these data establish a pivotal role for oxidative stress in systemic regulation of insulin and define a key role for the JunD protein in longevity.


Asunto(s)
Envejecimiento/fisiología , Insulina/metabolismo , Neovascularización Patológica/etiología , Estrés Oxidativo/fisiología , Páncreas/irrigación sanguínea , Animales , Antioxidantes/administración & dosificación , Antioxidantes/farmacología , Hipoglucemia , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal
8.
J Neurochem ; 123(3): 342-8, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22924626

RESUMEN

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS-specific GDH-null mice (CnsGlud1(-/-)); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns-Glud1(-/-) brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns-Glud1(-/-) mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns-Glud1(-/-) mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up-regulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.


Asunto(s)
Encéfalo/enzimología , Eliminación de Gen , Ácido Glutámico/metabolismo , Receptores de Glutamato/deficiencia , Receptores de Glutamato/genética , Transmisión Sináptica/genética , Animales , Encéfalo/patología , Encéfalo/fisiología , Células Cultivadas , Femenino , Glutamato Deshidrogenasa , Glutamina/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Técnicas de Cultivo de Órganos , Receptores de Glutamato/fisiología , Transmisión Sináptica/fisiología
9.
Proc Natl Acad Sci U S A ; 104(48): 19085-90, 2007 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-18006654

RESUMEN

Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting beta-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1beta and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.


Asunto(s)
Diabetes Mellitus Experimental/inmunología , Canales Iónicos/fisiología , Islotes Pancreáticos/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Mitocondriales/fisiología , Animales , Glucemia/análisis , Células Cultivadas/efectos de los fármacos , Citocinas/metabolismo , Diabetes Mellitus Experimental/patología , Progresión de la Enfermedad , Inflamación , Interferón gamma/farmacología , Canales Iónicos/deficiencia , Canales Iónicos/genética , Islotes Pancreáticos/patología , Lipopolisacáridos/farmacología , Linfocitos/inmunología , Linfocitos/patología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/fisiología , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Estreptozocina/administración & dosificación , Estreptozocina/toxicidad , Proteína Desacopladora 2
10.
Diabetes ; 67(10): 1949-1961, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30002133

RESUMEN

Ammonia detoxification and gluconeogenesis are major hepatic functions mutually connected through amino acid metabolism. The liver is rich in glutamate dehydrogenase (GDH) that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia, thus bridging amino acid-to-glucose pathways. Here we generated inducible liver-specific GDH-knockout mice (HepGlud1-/- ) to explore the role of hepatic GDH on metabolic homeostasis. Investigation of nitrogen metabolism revealed altered ammonia homeostasis in HepGlud1-/- mice characterized by increased circulating ammonia associated with reduced detoxification process into urea. The abrogation of hepatic GDH also modified energy homeostasis. In the fasting state, HepGlud1-/- mice could barely produce glucose in response to alanine due to impaired liver gluconeogenesis. Compared with control mice, lipid consumption in HepGlud1-/- mice was favored over carbohydrates as a compensatory energy fuel. The changes in energy partitioning induced by the lack of liver GDH modified the circadian rhythm of food intake. Overall, this study demonstrates the central role of hepatic GDH as a major regulator for the maintenance of ammonia and whole-body energy homeostasis.


Asunto(s)
Amoníaco/metabolismo , Gluconeogénesis/fisiología , Glutamato Deshidrogenasa/metabolismo , Hígado/metabolismo , Animales , Femenino , Gluconeogénesis/genética , Homeostasis/genética , Homeostasis/fisiología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo
11.
J Cereb Blood Flow Metab ; 38(10): 1754-1768, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-28621566

RESUMEN

The enzyme glutamate dehydrogenase (GDH; Glud1) catalyzes the (reversible) oxidative deamination of glutamate to α-ketoglutarate accompanied by a reduction of NAD+ to NADH. GDH connects amino acid, carbohydrate, neurotransmitter and oxidative energy metabolism. Glutamine is a neurotransmitter precursor used by neurons to sustain the pool of glutamate, but glutamine is also vividly oxidized for support of energy metabolism. This study investigates the role of GDH in neuronal metabolism by employing the Cns- Glud1-/- mouse, lacking GDH in the brain (GDH KO) and metabolic mapping using 13C-labelled glutamine and glucose. We observed a severely reduced oxidative glutamine metabolism during glucose deprivation in synaptosomes and cultured neurons not expressing GDH. In contrast, in the presence of glucose, glutamine metabolism was not affected by the lack of GDH expression. Respiration fuelled by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during an elevated energy demand, and it may reflect the large allosteric activation of GDH by ADP.


Asunto(s)
Metabolismo Energético/fisiología , Glutamato Deshidrogenasa/metabolismo , Glutamina/metabolismo , Neuronas/metabolismo , Animales , Respiración de la Célula/fisiología , Ácido Glutámico/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo
12.
Front Biosci ; 12: 1586-93, 2007 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-17127405

RESUMEN

We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background. The transgene expression was driven by either a 4000 bp or a 353 bp promoter. These transgenic lines, designated mIKO:INS4000 and mIKO:INS353, were viable and fertile. Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood. Total pancreatic insulin stores in mIKO:INS4000 and mIKO:INS353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice. Morphometric analysis of pancreas did not show any compensatory beta-cell hyperplasia. The majority of animals in both lines remained normoglycemic throughout their lives. Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS4000 male mice, likely due to impaired insulin secretion detected in those animals. In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features. Diabetes was never seen in female animals. The diabetes developed by mIKO:INS353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS4000 diabetic animals could live for several months. This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.


Asunto(s)
Diabetes Mellitus/genética , Insulina/genética , Animales , Animales Modificados Genéticamente , Glucemia/análisis , Diabetes Mellitus/sangre , Diabetes Mellitus/patología , Femenino , Expresión Génica , Intolerancia a la Glucosa/genética , Humanos , Insulina/biosíntesis , Insulina/sangre , Masculino , Ratones , Páncreas/patología
13.
Redox Biol ; 13: 541-549, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28755631

RESUMEN

Deterioration of pancreatic beta-cells plays a critical role in the development of type 2 diabetes. Among the various stressors contributing to these deleterious effects, glucotoxicity and superoxides have been proposed as major players. In this context, the mitochondrial uncoupling protein UCP2 is regularly associated with the stress response. In the present study, we tested the effects of UCP2 upregulation in mouse islets with beta-cell specific overexpression of UCP2 (RIP-UCP2). Islets were subjected to both chronic glucotoxicity (7 days at 30mM glucose) and acute oxidative stress (200µM H2O2 for 10min). Increased UCP2 expression did not alter mitochondrial potential and ATP generation but protected against glucotoxic effects. Glucose-stimulated insulin secretion was altered by both glucotoxicity and oxidative stress, in particular through higher basal insulin release at non-stimulatory glucose concentrations. The secretory response to glucose stimulation was partially preserved in beta-cells overexpressing UCP2. The higher rate of cell death induced by chronic high glucose exposure was lower in RIP-UCP2 islets. Finally, superoxide production was reduced by high glucose, both under acute and chronic conditions, and not modified by UCP2 overexpression. In conclusion, upregulation of UCP2 conferred protective effects to the stressed beta-cell through mechanisms not directly associated with superoxide production.


Asunto(s)
Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Estrés Oxidativo , Proteína Desacopladora 2/genética , Adenosina Trifosfato/metabolismo , Animales , Muerte Celular , Células Cultivadas , Glucosa/farmacología , Glucosa/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Proteína Desacopladora 2/metabolismo , Regulación hacia Arriba
14.
Cell Rep ; 13(2): 365-75, 2015 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-26440896

RESUMEN

Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.


Asunto(s)
Metabolismo Energético , Ácido Glutámico/metabolismo , Hipotálamo/metabolismo , Receptores de Glutamato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Glucosa/metabolismo , Glutamato Deshidrogenasa , Hipotálamo/citología , Hígado/metabolismo , Masculino , Ratones , Oxidación-Reducción , Receptores de Glutamato/genética
15.
Toxicol Lett ; 230(2): 198-207, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24583044

RESUMEN

The present study was designed to investigate the association between levels of organochlorine pesticides (OCPs) and liver enzyme responses in Cyprinus carpio. Fish were caught at three stations in the Büyük Menderes River (BMR): the origin, the Sarayköy station, and the estuary. Seventeen OCPs were quantified in liver tissue, as well as in river water by gas chromatography (GC)-electron capture detection, and structures were confirmed by negative chemical ionization-GC-mass spectrometry. The activities of CYP1A, GST, Se-GPx, CAT, and SODs were determined by spectrophotometry or fluorimetry. The mRNA levels of CYP1A, GST, and SOD1 were quantified by real-time RT-PCR. CYP1A and antioxidant enzyme activities were dramatically higher at the Sarayköy station, where OCP pollution is higher than the other two stations. Mn-SOD is responsible for the increase in total SOD activity in the Sarayköy samples. However, gene expression levels of certain enzymes were heavily suppressed. Our findings show that the transcriptional and functional responses of CYP1A and antioxidant enzymes are inversely correlated.


Asunto(s)
Hidrocarburos Clorados/toxicidad , Hígado/efectos de los fármacos , Plaguicidas/toxicidad , Superóxido Dismutasa/genética , Animales , Carpas , Citocromo P-450 CYP1A1/genética , Glutatión Transferasa/genética , Hígado/enzimología , ARN Mensajero/análisis
16.
Neurochem Int ; 59(4): 510-7, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21600947

RESUMEN

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme linking the Krebs cycle to the multifunctional amino acid glutamate. Thereby, GDH plays a pivotal role between carbohydrate and protein metabolisms, controlling production and consumption of the messenger molecule glutamate in neuroendocrine cells. GDH activity is under the control of several regulators, conferring to this enzyme energy-sensor property. Indeed, GDH directly depends on the provision of the co-factor NADH/NAD(+), rendering the enzyme sensitive to the redox status of the cell. Moreover, GDH is allosterically regulated by GTP and ADP. GDH is also regulated by ADP-ribosylation, mediated by a member of the energy-sensor family sirtuins, namely SIRT4. In the brain, GDH ensures the cycling of the neurotransmitter glutamate between neurons and astrocytes. GDH also controls ammonia metabolism and detoxification, mainly in the liver and kidney. In pancreatic ß-cells, the importance of GDH as a key enzyme in the regulation of insulin secretion is now well established. Inhibition of GDH activity decreases insulin release, while activating mutations are associated with a hyperinsulinism syndrome. Although GDH enzyme catalyzes the same reaction in every tissue, its function regarding metabolic homeostasis varies greatly according to specific organs. In this review, we will discuss specificities of GDH regulation in neuroendocrine cells, in particular pancreatic islets and central nervous system.


Asunto(s)
Sistema Nervioso Central/enzimología , Metabolismo Energético/fisiología , Glutamato Deshidrogenasa/metabolismo , Homeostasis/fisiología , Islotes Pancreáticos/enzimología , Animales , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/fisiología , Humanos , Mitocondrias/metabolismo
17.
Endocrinology ; 152(8): 3018-29, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21673098

RESUMEN

Inhibition of dipeptidyl peptidase-4 (DPP-4) activity improves glucose homeostasis through a mode of action related to the stabilization of the active forms of DPP-4-sensitive hormones such as the incretins that enhance glucose-induced insulin secretion. However, the DPP-4 enzyme is highly expressed on the surface of intestinal epithelial cells; hence, the role of intestinal vs. systemic DPP-4 remains unclear. To analyze mechanisms through which the DPP-4 inhibitor sitagliptin regulates glycemia in mice, we administered low oral doses of the DPP-4 inhibitor sitagliptin that selectively reduced DPP-4 activity in the intestine. Glp1r(-/-) and Gipr(-/-) mice were studied and glucagon-like peptide (GLP)-1 receptor (GLP-1R) signaling was blocked by an i.v. infusion of the corresponding receptor antagonist exendin (9-39). The role of the dipeptides His-Ala and Tyr-Ala as DPP-4-generated GLP-1 and glucose-dependent insulinotropic peptide (GIP) degradation products was studied in vivo and in vitro on isolated islets. We demonstrate that very low doses of oral sitagliptin improve glucose tolerance and plasma insulin levels with selective reduction of intestinal but not systemic DPP-4 activity. The glucoregulatory action of sitagliptin was associated with increased vagus nerve activity and was diminished in wild-type mice treated with the GLP-1R antagonist exendin (9-39) and in Glp1r(-/-) and Gipr(-/-) mice. Furthermore, the dipeptides liberated from GLP-1 (His-Ala) and GIP (Tyr-Ala) deteriorated glucose tolerance, reduced insulin, and increased portal glucagon levels. The predominant mechanism through which DPP-4 inhibitors regulate glycemia involves local inhibition of intestinal DPP-4 activity, activation of incretin receptors, reduced liberation of bioactive dipeptides, and activation of the gut-to-pancreas neural axis.


Asunto(s)
Glucemia/análisis , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Pirazinas/farmacología , Triazoles/farmacología , Adulto , Animales , Dipéptidos/farmacología , Dipeptidil Peptidasa 4/fisiología , Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores de la Hormona Gastrointestinal/fisiología , Receptores de Glucagón/fisiología , Fosfato de Sitagliptina , Nervio Vago/fisiología
18.
Islets ; 2(2): 124-6, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21099305

RESUMEN

The concept of functional heterogeneity among ß-cells proposes that each cell differs in its sensitivity to glucose and is recruited in a glucose-dependent manner into both biosynthetic and secretory active states in order to adapt insulin secretion to the metabolic environment. Therefore, characterization of ß-cell populations with different metabolic sensitivities would lead to the development of new therapeutic strategies. Based on heterogeneous surface PSA-NCAM expression on ß-cells, we have recently characterized two groups of cells, namely ß(high) and ß(low)-cells, in rat. Differences in insulin secretory capacity and in gene expression profiles suggest that ß(low)-cells are immature and/or non-functional cells in contrast to highly glucose responsive fully functional ß(high)-cells. Moreover, the relative distribution of ß(high) and ß(low)-cells correlated with physiological and pathological states regarding the functional ß-cell mass. Here we summarize our main results on ß(high) and ß(low)-cell populations and discuss some of the open remaining questions.


Asunto(s)
Células Secretoras de Insulina/citología , Células Secretoras de Insulina/fisiología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/análisis , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Variaciones Dependientes del Observador , Ratas , Ácidos Siálicos/análisis , Ácidos Siálicos/genética , Ácidos Siálicos/metabolismo
19.
Diabetes ; 58(3): 673-81, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19073765

RESUMEN

OBJECTIVE: Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion. RESEARCH DESIGN AND METHODS: ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging. RESULTS: Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of beta-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches. CONCLUSIONS: Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.


Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Mitocondrias/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Cromanos/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Masculino , Mitocondrias/efectos de los fármacos , NAD/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Tapsigargina/farmacología
20.
PLoS One ; 4(5): e5555, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19440374

RESUMEN

BACKGROUND: The mass of pancreatic beta-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous beta-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of beta-cells and investigated their physiological relevance in increased insulin demand conditions in rats. METHODS: Two rat beta-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. beta(high) and beta(low)-cells. Insulin release, Ca(2+) movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, beta(high) and beta(low)-cell distribution and functionality were investigated in animal models with decreased or increased beta-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat. RESULTS: We show that beta-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike beta(low)-cells, beta(high)-cells express functional beta-cell markers and are highly responsive to various insulin secretagogues. Whereas beta(low)-cells represent the main population in diabetic pancreas, an increase in beta(high)-cells is associated with gain of function that follows sustained glucose overload. CONCLUSION: Our data show that a functional heterogeneity of beta-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in beta-cell defects in type 2 diabetes.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Ácidos Siálicos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Arginina/farmacología , Western Blotting , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Citometría de Flujo , Glucosa/farmacología , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Leucina/farmacología , Masculino , Microscopía Confocal , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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